Saccharomyces cerevisiae using analysis of 3D reconstructions. We show that many of these results can be reproduced in sparse reconstructions, generated from a small fraction of the experimental data (5% of the data), and study the properties of such models. Finally, we propose for the first time a novel approach for improving the accuracy of 3D reconstructions by introducing additional predicted physical interactions to the model, based on orthologous interactions in an evolutionary-related organism and based on predicted functional interactions between genes. We demonstrate that this approach indeed leads to the reconstruction of improved models.The study of the 3D architecture of chromosomes has been advancing rapidly in recent years. While a number of methods for 3D reconstruction of genomic models based on Hi-C data were proposed, most of the analyses in the field have been performed on different 3D representation forms (such as graphs). Here, we reproduce most of the previous results on the 3D genomic organization of the eukaryote  Saccharomyces cerevisiae using analysis of 3D reconstructions. We show that many of these results can be reproduced in sparse reconstructions, generated from a small fraction of the experimental data (5% of the data), and study the properties of such models. Finally, we propose for the first time a novel approach for improving the accuracy of 3D reconstructions by introducing additional predicted physical interactions to the model, based on orthologous interactions in a different organism and based on predicted functional interactions between genes. Our proposed approach can facilitate future studies of 3D genome organization via improved models.Understanding the importance of genome architecture, the arrangement of genes within the genome and how this organization evolved has been intensively studied in recent years. Despite rapid progress in the field, accurate 3D modeling of genome organization remains a challenge. While a number of methods for 3D reconstruction of genomic models based on genome-wide experimental data were proposed, most of the analyses in the field have been performed on different 3D representation forms (such as graphs). Here, we reproduce most of the previous results on the 3D genome organization of the eukaryote  Understanding the importance of genome architecture, the arrangement of genes within the genome, and how this organization evolved has been intensively studied in recent years –4. It hai.e., the number of times each pair of genomic loci was observed in proximity—are the raw variables measured in Hi-C experiments, and provide a way to assess the co-localization of sets of loci in the nucleus. Indeed, many of the published results in the field are based on direct analysis of contact frequencies [Saccharomyces cerevisiae has suggested that many earlier results—including features of the contact maps, the co-localization of early firing replication origins and genomic location of tRNA genes—can be explained by random configurations of chromosomes that are tethered to a number of sites in the nucleus [Different methods have been applied and developed in recent years for the analysis of Hi-C data. Contact frequencies—quencies –10. Hi-Cquencies ,11–13. FThere have been a number of attempts to interpret Hi-C data by generating non-random 3D reconstructions based on distance constraints obtained from contact frequency maps ,9,14,15.S. cerevisiae genome, showing that previous results can be reliably reproduced in 3D models. We quantify the redundancy in information in the previously generated S. cerevisiae Hi-C map [S. pombe as well as predicted spatial interactions.Here we carry out a detailed analysis of the properties of populations of 3D reconstructions of the Hi-C map , showinget al. [S. cerevisiae genome et al. [SS. cerevisiae Hi-C map . Second, the optimization process was initialized randomly 4 times for each interaction set / sample and the best solution (out of 4) in terms of optimization error was selected. We studied the similarity of the models generated in this manner . To thiset al., based on a different modeling approach, identified inter-chromosomal contact enrichment between telomeres [Centromeres  were sigelomeres . It is pelomeres  Fig 2E)et al., bet al. [We observed that early-firing replication origins (Clb5-independent)  were co-et al. . We contIrr1p, part of the cohesin complex, were co-localized . For example, if the co-localization of TFs was similar for all models with sparseness of 0.5% and above, we concluded that the minimal sparseness required to reproduce this result was 0.5% see . We repeThese results may indicate that the scale of the Hi-C experiment could be reduced if 3D reconstruction is employed to analyze the data. We attempted to test this hypothesis by repeating the sparse reconstruction process with a modified approach, aiming to simulate a smaller experiment. In this scheme, rather than randomly sampling from the complete map of distance constraints, we gradually damped the number of observed reads (frequency) between each pair of DNA fragments in the experiment. We then proceeded with the reconstruction, regarding the input Hi-C map as a complete new dataset  scored higher than the baseline 0.5% models: The addition of 2,751 (orto-Hi-C 0.5%) interactions significantly improved (Wilcoxon one-tail signed rank at 0.01 significance level) on 0.5% Hi-C models in 12 out of 15 quality tests, compared with random models that significantly improved the reconstructions in none of the tests  significantly improved on 0.5%-HiC in 12 quality tests while random interactions improved in none of them (orto-Hi-C 5% vs. random p<10–4). We note that the latter orto-Hi-C models performed even better on 10 quality tests than models comprising of the complete dataset.We generated 20 models based on 0.5% of the Hi-C map with the addition of an equal number of orthologous Hi-C measurements, and 20 models with 10-fold the number of interactions (5%) transformed from i-C maps . In addii-C maps . We theni-C maps . By perforto-HiC models by the objective of random models generated from the same number of interactions, sampled from shuffled Hi-C maps and shuffled orthologous-Hi-C maps. We observed that the normalized objective function value is smaller than random (<1) for orto-Hi-C models, but increases with the number of added orthologous interactions . However, there was no improvement in the signal for telomeres, which moved away from the nuclear periphery towards the center of the nucleus (5%<10–3).Centromeres were significantly more co-localized in integrated models ( nucleus . This is–4 for each test). Improved results were seen also for all TFs around the TSS  and particularly Cohesin Irr1p , as well as correlation with PPI distance (–3). Moreover, results that were expected, but not significant, in Hi-C reconstructions, improved considerably, such as GO terms co-localization (–4), correlation with the co-expression coefficient of genes (–4) and protein abundance (PA) distance between genes (–4). We note that, in general, the signal increased with the addition of more orthologous-interactions to the models in the aforementioned tests.The most apparent improvement was found in functional model features, such as early- and late- firing replication origins Fig  and 4G, distance , p<10–3.lization ; p<10–4,of genes ; p<10–4 en genes ; p<10–4.et al. [We repeated the above process while integrating orthologous-interactions into other baseline models. 5% sparse Hi-C models  showed set al.  S6 Fig)et al. [SS. cerevisiae  . Given asee also , as wellS. cerevisiae Hi-C distance map, transforming them to estimated nanometric distances (CUFS 0.5%) interactions and observed that the addition improved results compared to the baseline model in 14 out of 15 quality tests  while the corresponding random models significantly improved on the baseline results in none of the tests  interactions significantly improved the results in 12 quality tests while the corresponding random models significantly improved in none of them (CUFS-5% vs. random p<10–4). We note that CUFS-5% showed stronger signals than the ones observed in Hi-C 100% on 9 quality tests, despite being based on considerably sparser data.We normalized CUFS distances according to the istances  and addeistances . We gene–3). Centromeres were marginally more co-localized in CUFS models compared to the baseline Hi-C models (5% = 0.044), while telomeres occupied regions closer to the nuclear envelope . Many functional sets were significantly more co-localized in CUFS-integrated models compared with the baseline Hi-C model, such as early- (–3), evolutionary breakpoints (–3), tRNA clusters (–3). Global TFs co-localization (0.5% = 0.0175) but not for 5% models, however Irr1p bound genes  were significantly more co-localized than before. Other functionally related features that improved include GO terms (–4), as well as the correlation between pairwise gene distances and co-expression coefficient (–3), PPI distances (–4) and similarity in expression levels (–4). We repeated the above process while integrating CUFS-interactions into other baseline models, as was done for orthologous-interactions, with comparable results , but still higher than the baseline . Model senvelope ; p<0.01.s early- ; p<0.01 s early- ; p<10–3,akpoints ; p<10–3,clusters ; p<10–3.lization  was imprGO terms ; p<10–4,fficient ; p<10–3,istances ; p<10–4 n levels ; p<10–4. results –S8 Figs.S. cerevisiae is great; thus, if we aimed at studying 3D reconstructions we could do it with significantly sparser amount of data. We were able to reproduce many of the previously reported results on yeast genomic organization for the first time in 3D reconstructions. We also suggest that distances on a protein-protein interactions graph are correlated with gene pairwise distances on the genomic models. In addition, 3D reconstructions can be improved using our proposed approach to generate models that reproduce previous results better. We applied the proposed method to several baseline models and demonstrated significant improvement on a set of quality tests. The ability to improve models which were based on the complete Hi-C dataset suggests that our predicted distances may contain valuable information that is complementary to the S. cerevisiae Hi-C dataset. Our results support previous observations that the genomic organization of genes is partially conserved between species [e.g., gene expression levels, protein-protein interactions as well as metabolic networks. Improvement of 3D reconstructions can be further extended to integrate several Hi-C datasets from the same organism, where available. In multicellular organisms, tissue-specific data may be employed to improve the reconstruction, such as a list of known active / inactive genes in a cell type, condition, or developmental stage. Hi-C data from different tissues can also potentially be utilized and integrated into the model; however, in this case attention should be given to choosing tissue-invariant features, such as some aspects related to the local chromosome folding of topologically associated domains (TADs) [In this study, we proposed an approach for the reconstruction of 3D genomic models. Our conclusions are that at least according the current quality tests the redundancy in Hi-C measurements in  species . These r species . The pros (TADs) ,36.There are a number of computational challenges related to 3D reconstruction in higher eukaryotes. First, whole-genome reconstructions have been limited to small genomes due to computational costs . It is ppopulation of 3D structures. The latter issue can be partially dealt with by producing a population of reconstructions [Finally, most Hi-C experiments to date were carried on cell populations, thus measuring the average contact frequencies of a ructions ,15,18, aS. cerevisiae (S288c) and S. pombe (972h) genomes were obtained from NCBI, as well as the coding sequences of their 5,888 (SC) and 5,123 (SP) protein-coding genes.Sequences of the Hi-C data was obtained from . Hi-C mapi,qi are a pair of beads in the model and δi is their expected distance (the input to the program). The solution is bounded, to comply with the following constraints [http://www.cs.tau.ac.il/~tamirtul/reconstruction.zip3D reconstructions were generated using a modified version of the program provided by . The prostraints : nucleusstraints . The recSparse models were generated by uniformly sampling a portion of the set of Hi-C interactions . Hi-C interactions were binned at a resolution of 10kbp prior to sampling. Sampling was repeated independently 20 times (generating 20 sets of interactions), from each set 4 models were constructed . The best model out of the 4 in terms of optimization error  was chosen to represent the set of sampled interactions, in order to avoid solutions that converged to local minima. For the sake of consistency, the 80 models generated from the 100% map were partitioned into 20 groups and selected according to the same principle.e.g., 20); (2) filtering reads below 1 (a detection threshold); (3) quantizing the reads by rounding them to the nearest integer; (4) summing the reads per 10kbp bin; (5) converting the observed contact frequencies to distances according to rank, i.e. by assigning the highest value in the damped-map with the lowest value in the distribution of Hi-C distances and so on, until the sparse damped-map was completely converted.In S. pombe were obtained from [S. pombe pair of genes was assigned with a spatial distance according to the nearest measured coordinates on the map. Next, 3,367 orthologous families obtained from the manually curated orthologs database at PomBase [S. pombe genes to their identified respective orthologs in S. cerevisiae (averaged on multiple genes). So that, given a distance matrix DB in organism B, the orthologous-transformed matrix in organism A is given by:Oj is the set of orthologous genes in organism B corresponding to gene j in organism A. 3D distances were further normalized to account for the different dimensions of the nucleus in the 2 organisms, by scaling S. pombe distances to have the same median as the S. cerevisiae set of Hi-C distances , after excluding bins with existing Hi-C interactions see . The samPoor-get-richer sampling:Data: distance matrix C.Result: distance matrix C containing additional n interactions.Init: compute the degree of each node according to:N{} indicates the number of elements satisfying the condition. A non-zero degree was assigned for beads with no interactions, for numerical stability.begin if distance already known foreach sample up to nRemove orthologous interactions  with probability:Draw from the available orthologous interactions Update node degrees.S. cerevisiae ORFs. The CUFS distance between a pair of genes, given their two codon frequency vectors, p and q , was then computed by the Endres-Schindelin metric for probability distributions [dKL is the Kullback—Leibler divergence. In order to transform CUFS distances to nanometric distances independently of additional data, we used a simple linear model. CUFS distances were converted to nanometric distances by scaling the map to have the same median as the Hi-C distances. The top and bottom 10% of distances were considered for integration into the Hi-C distance map (The codon usage frequency similarity (CUFS)  was usedibutions :dKL by averaging the distances in each bin. CUFS interactions were sampled to produce an additional set of interactions added to Hi-C interactions see , after e4 μm2, random: 8.9x104 μm2). The magnitude of this error is related to the number of interactions in the input matrix, which was identical for random and real. When testing for co-localization of sets of elements in random models, we used their original position on the chromosome (rather than their position on the permuted coordinates).Random interactions were obtained by permuting the coordinates of the 10kbp-binned Hi-C interaction map. Thus, the permuted maps preserve the distribution of distances in the dataset. 20 permutations were drawn, and the complete permuted maps were utilized to generate the random reconstructions that appear in In addition, random sets were sampled and added to a subsampled, non-permuted Hi-C distance map using poor-get-richer sampling see , after eCo-localization / dispersion analysis of sets of loci / genes was performed using a normalized set distance (NSD) measure. NSD is defined as the mean set distance for the set of interest, normalized by the expected distance of random sets of equal size drawn from the same model. Specifically, 100 samples were drawn from all possible gene pairs, for gene sets; or samples from a uniform 10kbp coordinate grid on the model, for other sets of loci (such as early replication origins). Thus, the obtained value is <1 for co-localized sets and >1 for dispersed sets.et al.) and long arms (>250kbp) (short-vs-long); second, the ratio of the average distance between the two arms of each chromosome and the rest of the arms (cis-vs-trans); third, the ratio of the average distance between regions on the same arm and their distance from other arms (self-vs-else). All distances along the arms were sampled in 10kbp resolution.Chromosome arms analysis followed that of  and provModel similarity quantifies how similar repeated reconstructions are according to pairwise distances. Model reconstruction is stochastic due to both random initialization of the reconstruction program, as well as random sampling of interaction sets. Model similarity was estimated by computing the correlation between pairwise distances in 10kbp resolution between all generated models in the same category. For example, a total of 80 models were generated in the Hi-C-0.5% category (4 instances from 20 sampled sets of interactions). For each of the 80x80 possible pairs of models the pairwise distances between 10kbp-spaced genomic loci were utilized to compute Spearman’s rank correlation between them. Similarity was defined as the average correlation coefficient between all pairs.p-q|/(p+q), see Figs Spearman’s rank correlation with CUFS, protein abundance ,44 distae.g., HiC-0.5% vs. an orthologous-integrated model). We chose a paired-test in order to compare each set of sampled Hi-C interactions to its extended set .P-values, unless stated otherwise, were computed using a one-tailed Wilcoxon signed rank test, comparing the distributions of values obtained for 2 model types  was smaller than 0.01 we rejected the null hypothesis that the observed improvement on the tests was a random effect.In order to estimate the probability of observing the resultant improvement on tests in integrated models by chance, we shuffled the models between two categories—Irr1p co-localization was computed by averaging the result over the 3 libraries. Co-expression correlation coefficients were computed between the mRNA expression profiles of genes in 530 conditions [Centromere positions were obtained from . Telomernditions . Full GOnditions  and mappnditions  appearinnditions –51 and fWe performed hierarchical clustering see  by utiliS1 Fig(A)-(D) Reconstructed models for different levels of sparseness. (E) A random model. (F)-(G) Integrated models, containing predicted distances in addition to the complete Hi-C dataset. (H) An illustration of the proposed reconstruction approach employed in (F)-(G).Models are not illustrated in precise, equal scale. (EPS)Click here for additional data file.S2 Fig(A) Optimization objective function (squared error between input distances and resultant model). (B) Mean objective per input constraint. (C) Ratio of the average distance between points on a chromosome arm and the average distance between that arm and the rest of the chromosome arms. (D) Ratio of the average distance between short chromosome arms (<250kbp) and long chromosome arms (>250kbp). (E) Co-localization results for TF binding sites in the upstream activation sequence (UAS) library. (F) Co-localization results for TF binding sites in the open reading frame (ORF) library. (G) Co-localization results of previously identified GO terms (details in (H) Spearman's correlation between pairwise distances of genes and their Gene Ontology terminology distance  Spearman's correlation between pairwise distances of genes and their average protein abundance .(EPS)Click here for additional data file.S3 FigThe figure shows the pairwise distance heat map between telomeres, averaged across 80 reconstructions, and ordered by utilizing hierarchical clustering on the Hi-C 100% model. Telomeres from the long chromosome arm are denoted with L while ones from the short arm are denoted with S. Additional matrices corresponding to Hi-C 5%  and random models  are presented for reference. The largest cluster (1) comprises of 15 telomeres from 11 chromosomes . Additional clusters appear along the diagonal. In addition, it can be seen that the 2 telomeres of each chromosome tend to interact. Distances are in micrometers.(EPS)Click here for additional data file.S4 FigThe results in this figure mirror those appearing in (EPS)Click here for additional data file.S5 FigThe results in this figure mirror those appearing in Figs (EPS)Click here for additional data file.S6 FigThe results in this figure mirror those appearing in Figs (EPS)Click here for additional data file.S7 FigThe results in this figure mirror those appearing in Figs (EPS)Click here for additional data file.S8 FigThe results in this figure mirror those appearing in Figs (EPS)Click here for additional data file.S9 FigThis figure presents the histogram of distances in the Hi-C distance map (100 bins).(EPS)Click here for additional data file.S10 FigS. pombe distance map (100 bins), after transformation to S. cerevisiae coordinates (orthologous genes) and scaling. For the purpose of reconstruction, distances up to the marked threshold were sampled. These distances are expected to be more reliable. Note that the median is equal to the one in This figure presents the histogram of the normalized (EPS)Click here for additional data file.S11 FigThis figure presents the histogram of the scaled CUFS distance map (100 bins). For the purpose of reconstruction, distances below the left marked threshold and above the right threshold were sampled. Note that the median is equal to the one in (EPS)Click here for additional data file.


Furthermore, the pro-survival and pro-migratory effects of SCF were augmented by c-kit overexpression and abrogated by c-kit inhibition with imatinib. Mechanistically, c-kit activation in CPCs led to activation of the PI3K and the MAPK pathways. With the use of specific inhibitors, we confirmed that the SCF/c-kit-dependent survival and chemotaxis of CPCs are dependent on both pathways. Taken together, our findings suggest that c-kit promotes the survival/growth and migration of human CPCs cultured ex vivo via the activation of PI3K and MAPK pathways. These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit.A recent phase I clinical trial (SCIPIO) has shown that autologous c-kit+ cardiac progenitor cells (CPCs) improve cardiac function and quality of life when transplanted into patients with ischemic heart disease. Although c-kit is widely used as a marker of resident CPCs, its role in the regulation of the cellular characteristics of CPCs remains unknown. We hypothesized that c-kit plays a role in the survival, growth, and migration of CPCs. To test this hypothesis, human CPCs were grown under stress conditions in the presence or absence of SCF, and the effects of SCF-mediated activation of c-kit on CPC survival/growth and migration were measured. SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions. In addition, SCF significantly promoted CPC migration  The adult mammalian heart has long been considered a post-mitotic organ that is incapable of regeneration. This notion was challenged by several reports suggesting that the heart is not terminally differentiated and is capable of regeneration, albeit to a limited extent –3. Recenc-kit+ cells with properties of CPCs were first described in the rat heart by Beltrami et al. in 2003 . When isCellular Kit (c-kit)  encodes a 145 kDa transmembrane glycoprotein that belongs to the type-III receptor tyrosine kinase family, which includes the platelet-derived growth factor receptor and macrophage colony stimulating factor 1 receptor –23. StemAlthough c-kit has been widely used as a surface antigen for identification and isolation of resident CPCs, its role in regulating the properties of CPCs remains unclear. In the present study, we hypothesized that c-kit plays a role in regulating the growth, survival, and migration of human c-kit+ CPCs. Our results indicate that activation of c-kit by SCF promotes the growth, survival and migration of human CPCs, and that these effects are mediated by the PI3K-AKT and MEK-ERK pathways.Atrial appendages specimens were collected from patients during the coronary artery bypass graft performed at Jewish Hospital at University of Louisville. A written consent agreement was obtained for collection of discarded atrial appendages according to a protocol approved by the Institutional Review Board on human subject research at University of Louisville. c-kit+ human cardiac stem cells were isolated and cultured as previously described . WrittenTM (Invitrogen) was used to assess cell growth and viability. For manual cell counting, approximately 50,000 cells were plated per well of a 24-well plate. CPCs were then serum starved for 24 hr and treated with or without 100 ng/ml SCF in serum free media. For bFGF and VEGF treatments, CPCs were incubated with 50 ng/ml bFGF (Peprotech) or 20 ng/ml VEGF (Invitrogen) either alone or in combination with SCF as indicated. After 3 days of growth factor treatment, cells were trypsinized and counted using a hemacytometer. For measurement of cell viability using PrestoBlueTM, 10,000 CPCs were plated per well of a 96-well plate. When indicated, CPCs were treated with 0.5 μM imatinib mesylate  for 2 hr prior to SCF treatment. PrestoBlueTM cell viability assay was performed 72 hr post-growth factor treatment according to the manufacturer’s instructions. Briefly, the 10x reagent was mixed with an appropriate volume of serum free medium, added to the cells and incubated at 37°C for an hr. The viability was assessed by measuring the fluorescence at Ex/Em 560/590 nm.Manual cell counting or PrestoBlueApproximately 10,000 cells per well were plated on a 96-well tissue culture plate. Cells were serum-starved for 24 hr followed by treatment with 20 μM BrdU for 24 or 72 hr in the presence or absence of SCF in the assay medium. After BrdU labeling, cells were fixed and immunostained using anti-BrdU antibody  and Alexa555-conjugated secondary antibody . Images were captured using EVOS® FL Cell Imaging System (Life Technologies), and the BrdU-positive cells were counted manually.® Homogeneous Caspase-3/7 Assay kit (Promega) was used to measure the caspase activity in CPCs according to the manufacturer’s instructions. Briefly, approximately 10,000 CPCs were plated per well. The next day, the medium was changed to serum free media or same media containing SCF. After 3 days of SCF treatment, the caspase activity in the cells was measured by adding the pro-fluorescent substrate and reading the fluorescence at Ex/Em 499/521 nm. The values were normalized to the untreated control.Apo-ONETM. For H2O2 experiments, approximately 10,000 cells per well were plated on a 96-well tissue culture plate. On the next day, cells were treated with 100 ng/ml SCF or 10 μg/ml insulin (Gibco) in serum free media. On the following day, CPCs were subjected to 0.5 mM H2O2  treatment for 1 hr. Cell viability was assessed 3 days later using PrestoBlueTM.For DMNQ experiments, approximately 10,000 human c-kit+ CPCs per well were plated on a 96-well tissue culture plate. On the next day, cells were treated with 100 ng/ml SCF in serum free media. For the imatinib treatment groups, cells were treated with 0.5 μM imatinib for 2 hr prior to the SCF treatment. On the second day, CPCs were subjected to 8 μM DMNQ  treatment in the groups indicated. After 3 days of DMNQ treatment, cell viability was assessed using PrestoBlueCells were harvested with Laemlli buffer and heated for 10 minutes at 100°C. Protein concentration was estimated using bicinchoninic acid (BCA) assay kit purchased from Thermo scientific following manufacturer’s instructions. Before loading the samples on the gel, 3 μl of β-mercaptoethanol was added per 100 ul of sample and heated for 3 minutes at 100°C. Cell lysate (25 or 50 μg of protein) was loaded on 4–20% Tris-Glycine gel and separated by electrophoresis. The separated proteins were transferred to a PVDF membrane, blocked with 5% skim milk or 5% bovine serum albumin for 1 hr followed by probing with primary antibody overnight at 4°C. The blot was then incubated with HRP-conjugated secondary antibody for 1 hr and developed using Amersham ECL Prime Western Blotting Detection Reagent . Primary antibodies used were anti-c-kit , anti-phospho c-kit , anti-AKT , anti-phospho AKT , anti-ERK 1/2 , anti-phospho ERK 1/2 , and anti-α-tubulin .Boyden chambers containing transwells (24-well plate format) with 8 μm pore size were purchased from Corning. Typically, 30,000–40,000 cells were plated in the upper chamber of the transwell. After 24 hr, the medium in the upper chamber was changed to assay medium and bottom chamber was filled with the same medium alone or medium containing 100 ng/ml SCF. For inhibition of c-kit activity, cells were pre-treated with 0.5 μM imatinib for 2 hr prior to the SCF treatment. After 24 hr, the cells migrated to the lower surface of the transwell membrane were fixed with 3.7% formaldehyde for 10 minutes, and the cells still remaining on the upper chamber were scraped off. The fixed cells were then permeabilized with 0.25% Triton X-100 in PBS for 10 minutes followed by incubation with 50 μg/ml propidium iodide solution for 10–15 minutes. Images were acquired using EVOS® FL Cell Imaging System (Life Technologies), and the number of migrated cells was quantified using the NIS-Elements (version 4.2) software.TM Lentiviral Expression System and pLenti6-V5 vector (Invitrogen) according to the manufacturer’s instructions. Virus was concentrated using the Lenti-X Concentrator (Clontech) and was stored at -80°C until its use. On the day of the experiment, CPCs were transduced with complete medium containing either mCherry or c-kit virus and 6 μg/ml polybrene (Sigma). On the following day, the media was replaced with fresh complete media. Cells were cultured for 5 days to allow expression of mCherry or c-kit prior to being used for the experiments.The lentivirus carrying mCherry or FLAG-tagged murine c-kit was produced by using the ViraPowerWortmannin and PD98059 were purchased from Cell Signaling Technology and used at 200 nM and 40 μM concentrations, respectively. For the Western blot analysis, CPCs were serum starved for 24 hr and pre-treated with the inhibitors for 2 hr followed by 20 min of SCF (100 ng/ml) treatment. For the cell viability and migration assays, cells were pre-treated with the indicated inhibitors for 2 hr prior to being treated with SCF.i.p. 100 mg/kg). The excised hearts were washed in PBS, minced into small pieces and enzymatically dissociated with Collagenase II (5 μg/ml in PBS) (Worthington) with gentle agitation for 45 min in 37°C. After Collagenase II inactivation with DMEM/F12 medium containing 10% FBS, cells were centrifuged at 600 xg for 10 min. The cell pellet was suspended in growth medium consisting of DMEM/F12, 10% FBS, bFGF (10 ng/ml), EGF (10 ng/ml), LIF (10 ng/ml), ITS (Insuline/Transferrine/Selenium), glutamine and penicillin/streptomycin. The single cell suspension was plated in the tissue culture flask. After 4–6 days of expansion, c-kit+ cells were sorted with using MACS immunomagnetic labeling method. Briefly, cells were detached by enzymatic digestion with 0.25% trypsin, washed in PBS and stained with rat monoclonal antibody anti-mouse c-kit (clone 2B8) conjugated with FITC (eBioscience) for 30 minutes on ice. Subsequently, cells were washed in PBS and stained with magnetic beads directed anti-FITC (Miltenyi) for 30 minutes on ice. The c-kit cells were sorted by positive selection with using MS magnetic column. The sorted cells were analyzed for the purity with using flow cytometry . On average 85% purity was obtained from all isolated fractions. Sorted cells with purity below 80% were excluded from the experiments. For differentiation, c-kit+ CPCs were passaged at seven days post-sort and seeded in on the 12-well plate at density of 50,000 cells per well. When cells reached approximately 90% of confluence, they were stimulated with differentiation or control media  in the presence or absence of SCF (50 ng/ml). Cells were maintained for 4 weeks with media change once every two days. The myocyte, endothelial, and smooth muscle differentiation markers were evaluated at mRNA level by real time qPCR.Mice  were euthanized by sodium pentobarbital injection  or H cycling , whereasnd death . As showe stress . In conte stress . These rFor successful engraftment of the donor cells following transplantation, homing of the CPCs to the recipient myocardium and the injured area is required. SCF/c-kit signaling has been implicated in stimulating migration of various c-kit-expressing cell types , 49, 50.Activation of c-kit leads to subsequent activation of different downstream pathways, including the PI3K-AKT , 53, p38Next, we tested if c-kit activity has any significant effect on the ability of CPCs to differentiate into cardiac lineages. For this, we subjected murine c-kit+ CPCs to a dexamethasone-induced differentiation protocol and co-treated the cells with or without SCF. Dexamethasone was able to mostly induce markers of myocytes in CPCs . AlthougInitial attempts to treat ischemic cardiomyopathy with administration of autologous c-kit+ CPCs have yielded promising results in both animals and humans, many unanswered questions remain regarding the use of these cells for cardiac repair, and many hurdles need to be overcome to establish the efficacy of this new therapy . There iThe present study aimed at investigating the role of a cardiac stem/progenitor cell antigen, c-kit, in regulating the cellular properties of adult human cardiac c-kit+ CPCs. Our findings demonstrate, for the first time, that i) activation of c-kit leads to increased proliferation and survival and stimulates migration/chemotaxis in human CPCs, and ii) both the PI3K-AKT and MEK-ERK pathways are involved in mediating the salutary effects of SCF/c-kit signal in CPCs. These results imply that the SCF/c-kit signaling pathway in CPCs can be exploited to overcome one of the major problems in the current field of cardiac cell therapy: poor survival and engraftment of the transplanted cells.For successful cell therapy of ischemic cardiomyopathy, the injected cells must find their ways into the host myocardium and to the site of injury. Hence, homing and migration of these CPCs after transplantation are critical cellular properties that determine their therapeutic and regenerative activities. However, this poses a major problem especially when the cells are delivered via the intracoronary route, as cells must transverse through the endothelial barrier to enter into the interstitial space and migrate towards the infarcted area. In a recent study, we have shown that > 60% of the injected CPCs are lost during the first 5 min of intracoronary injection , suggestKitW/KitW-v) mouse [The SCF/c-kit pathway is often activated and plays an important role during injury in various tissues –64. For v) mouse , demonstv) mouse . These ov) mouse . Althougin situ by intramyocardial injection of SCF and potentiate the regenerative response in the aging murine heart [The functional importance of SCF/c-kit signaling in regulating c-kit+ CPCs and cardiac regeneration is further underscored by a recent study that demonstrated the benefits of SCF therapy. A study by Yaniz-Galende and colleagues showed that adenovirus-mediated gene delivery of the membrane-bound form of SCF to the myocardium can provide long-term improvement in cardiac structure and function in an animal model of myocardial infarction (MI) . The SCFne heart . The autne heart . These sOur present findings that c-kit activation elicits pro–growth and–migratory signals in human c-kit+ CPCs are consistent with these previous observations. As such, augmentation of the inherent c-kit activity in exogenous CPCs appears a viable option to further improve the outcome of CPC-based cardiac cell therapy. Additional studies using animal models of myocardial ischemia-reperfusion are required to test if such strategy is able to enhance post-transplantation survival and engraftment of the cells and ultimately augment the efficacy of cell therapy. Also, it will be of interest to study how differences in the percentage of c-kit-expressing cells or in the level of c-kit expression among patients translate into differences in the therapeutic efficacy of individual CPC populations.


Objective: To evaluate the mechanical property and biocompatibility of the Wnt pathway inhibitor (ICG-001) delivering collagen/poly(l-lactide-co-caprolactone) (P(LLA-CL)) scaffold for urethroplasty, and also the feasibility of inhibiting the extracellular matrix (ECM) expression in vitro and in vivo. Methods: ICG-001 (1 mg (2 mM)) was loaded into a (P(LLA-CL)) scaffold with the co-axial electrospinning technique. The characteristics of the mechanical property and drug release fashion of scaffolds were tested with a mechanical testing machine (Instron) and high-performance liquid chromatography (HPLC). Rabbit bladder epithelial cells and the dermal fibroblasts were isolated by enzymatic digestion method. -2,5-Diphenyltetrazolium Bromide (MTT) assay) and scanning electron microscopy (SEM) were used to evaluate the viability and proliferation of the cells on the scaffolds. Fibrolasts treated with TGF-β1 and ICG-001 released medium from scaffolds were used to evaluate the anti-fibrosis effect through immunofluorescence, real time PCR and western blot. Urethrography and histology were used to evaluate the efficacy of urethral implantation. Results: The scaffold delivering ICG-001 was fabricated, the fiber diameter and mechanical strength of scaffolds with inhibitor were comparable with the non-drug scaffold. The SEM and MTT assay showed no toxic effect of ICG-001 to the proliferation of epithelial cells on the collagen/P(LLA-CL) scaffold with ICG-001. After treatment with culture medium released from the drug-delivering scaffold, the expression of Collagen type 1, 3 and fibronectin of fibroblasts could be inhibited significantly at the mRNA and protein levels. In the results of urethrography, urethral strictures and fistulas were found in the rabbits treated with non-ICG-001 delivering scaffolds, but all the rabbits treated with ICG-001-delivering scaffolds showed wide caliber in urethras. Histology results showed less collagen but more smooth muscle and thicker epithelium in urethras repaired with ICG-001 delivering scaffolds. Conclusion: After loading with the Wnt signal pathway inhibitor ICG-001, the Collagen/P(LLA-CL) scaffold could facilitate a decrease in the ECM deposition of fibroblasts. The ICG-001 delivering Collagen/P(LLA-CL) nanofibrous scaffold seeded with epithelial cells has the potential to be a promising substitute material for urethroplasty. Longer follow-up study in larger animals is needed in the future. It has etc. .In spite of the rapid developments of the surgical procedures for urethroplasty, the recurrence rate was still high (approximately 20%) because of submucosa fibrosis and scar formation in the urethra after substitute surgery ,5. In reExcessively deposited ECM of urethral submucosa was believed to be closely related to urethral restructure . Signifietc. [etc. The Wnt pathway inhibiter ICG-001 we used here is a small molecule with 548.63 in molecular weight [It is known that the TGF-β pathway plays an important part in a variety of fibrotic diseases ,15,16, aetc. ,28,29,30etc. . Furtherr weight .l-lactide-co-caprolactone) (P(LLA-CL)), which morphologically and structurally mimicked the ECM of native tissue [l-lactic acid and ε-caprolactone, which possesses good biocompatibility, biological degradability and mechanical properties, but because of its hydrophobic characteristic, it is not conducive to cell adhesion and proliferation. In our study, collagen and P(LLA-CL) were combined to obtain good biocompatibility and mechanical strength of the desired nanofiber [Electrospinning is an adaptable method for fabrication of scaffolds . The scae tissue . Collageanofiber . Compareanofiber ,36.in vitro and in rabbits urethral defects model in order to provide preliminary evidence and foundation for the large animal study and clinical practice in the future.In this study, We combined the technique of co-axial electrospinning technology with the Wnt pathway inhibitor ICG-001 to produce a functional electrospun collagen/P(LLA-CL) nanofiber scaffold, then evaluated its anti-fibrosis effect The thickness of the non-drug scaffolds was 0.75 ± 0.16 mm and ICG-001  deliveriThe controlled release of ICG-001 from drug delivering collagen/P(LLA-CL) scaffolds were analyzed with High-performance liquid chromatography (HPLC) . The relSuccessful epithelial cell A and fib2. The structure with cells was cultured in D-KSFM for 1 week before being tested and implanted into the animal. Fibroblasts were cultured and passaged in six-well plates for the anti-fibrosis test.Cultured epithelial cells were seeded onto each scaffold with a density of 0.5 million/cmOn day three and seven, SEM was used to observe the growth of the cells on the scaffolds. The SEM showed that the epithelial cells attached well on both kinds of scaffolds on day three A,B. The MTT assay showed a similar rate of proliferation of epithelial cells on both non-drug scaffold and drug delivering scaffold. The proliferation of each scaffold could provide a feasible environment for the cells, the relative absorption between the third and seventh day was significantly different. However, the relative absorption between each kinds of scaffold was not statistically significant which demonstrated that the ICG-001 was not toxic to the epithelial cells .Fibroblasts were treated with TGF-β1 with or without ICG-001 for three days to evaluate the phenotype change of myofibroblasts and ECM expression. The signals of collagen type 1 and 3 was significantly reinforced in the TGF-β1 treated fibroblasts A,E. CollQuantitative real-time PCR was performed at day three to evaluate the change of gene expression of fibroblasts at the mRNA level . CollageWestern blot was performed for relative quantitative analysis of collagen type 1, collagen type 3, fibronectin and α-SMA A. The quTwelve rabbits in two groups underwent operations and survived within three months. Five out of six rabbits in group 1 developed narrow urethral lumens according to the urethrography C and oneIn histology test of group 1, the lumen surface formed discontinued epithelial layer according to the H&E staining A and AE1In the present study, it was demonstrated that the biocompatibility and mechanical properties of the ICG-001-delivering collagen/P(LLA-CL) electrospun scaffold was sufficient for bladder epithelial cells proliferation and could be applied to urethroplasty. The co-axial electrospun scaffold delivering ICG-001 delivered drug release in a controlled fashion and had obvious effects of anti-fibrosis.in vitro and in vivo [The ideal scaffolds could undertake many tasks such as to provide the necessary mechanical property and deliver inductive biomolecules ,38. Scaf in vivo ,43. As w in vivo . Therefoin vivo study in the future.The advantage of core-shell co-axial electrospinning secured a controlled release of nearly 75% ICG-001 in scaffolds as long as 30 days. According to the instruction of the manufacturer, the pharmaceutical activity of ICG-001 could be maintained three months at room temperature. It was reported that the urethral scar formed in the first month after urethral injury ,46, so tet al. demonstrated that the use of small molecule drugs to inhibit the Wnt pathway is an effective method for the treatment of fibrosis; it could obtain a similar effect of inhibiting the TGF-β pathway, but has good cell tolerance [in vitro. In the immunofluorescence studies, highest expression of collagen type 1 and 3 was noticed in the TGF-β1 treated group; when combined with ICG-001-released medium, the expression showed an obvious decline. This phenomenon was also found in the group with ICG-001-released medium alone compared with the control group.Wnt signaling has been implicated into the pathogenesis of various human disorders such as fibrotic diseases . Activatolerance . In the Real time PCR revealed the effects of ICG-001 at the RNA level. RNA expression of collagen type 1, collagen type 3, and α-SMA was significantly elevated in the groups with ICG-001-released medium compared with the TGF-β1 alone, and control group. α-SMA is a biomarker of myofibroblasts which demonstrated the phenotype change of fibroblasts and higher expression of the ECM gene . MyofibrWestern blot results demonstrated that the ICG-001-released solution could inhibit the production of ECM related protein at the protein level, including α-SMA, collagen type 1, 3 and fibronectin. The intensity of western blot bands in the ICG-001 group was decreased significantly compared with the TGF-β1 treated group and control group respectively. The results were consistent with that in the fluorescence and real-time PCR.in vitro study, we investigated the therapeutic effect of the ICG-001 delivering scaffold in the rabbit urethra defect model. Collagen deposition was inhibited significantly by the ICG-001 delivering scaffold according to the histology. ICG-001 could be released from the scaffold gradually after transplanting into the urethral defect to inhibit the inflammation and ECM deposition during the healing process of the urethra. In our previous meta-analysis about anti-fibrosis drugs for urethral stricture, the efficacy of various drugs were not yet verified [Based on verified . This fuIt was reported that the airway epithelial cell layer could be maintained by ICG-001 and that airway epithelial cell apoptosis was significantly decreased in bleomycin-induced animal models . This chl-lactide-co-caprolactone) (P(LLA-CL))  was provided by Nara medical university. Type I collagen was obtained from Sichuan Ming-Rang Bio-Tech Co., Ltd. . And 2,2,2-trifluoroethanol was from Fine chemicals, Shanghai, China. The ratio of collagen: P(LLA-CL) = 25:75. ICG-001 was purchased from Selleckchem .Poly(Collagen/P(LLA-CL) scaffolds delivering ICG-001  were conThe non-drug co-axial collagen/P(LLA-CL) electrospun nanofiber scaffold was fabricated as a control group, all the processes were same, but 60 µL DMSO without ICG-001 was added in the core solution.Scanning electron microscopy  was used to observe the morphology of the scaffolds. Specimens were punched into 1.2 cm-diameter disks and cryopreserved at −80 °C for 2 h, then freeze-dried overnight and preserved in a vacuum container. The specimens of scaffolds were imaged under SEM. Nanofiber diameter was measured with 200 fibers by image analysis software Image-J.To compare the mechanical properties of different scaffolds, small intestinal submucosa (SIS), a commercial biomaterial for urethra reconstruction, was used as the control material. Tensile strength was measured by an instron tensile tester . All samples were prepared as longitudinal strips (20 mm in length and 10 mm in width). Each sample of scaffolds was fixed onto the clamps and pulled at 5 mm/minute crosshead speed until rupture. Burst pressure was measured by gradually increasing hydrostatic pressure within the scaffolds at a rate of 80 mmHg/min. Then Bluehill software  was used to calculate the maximal tensile strength.The ICG-001 delivering scaffold was punched into dishes with 1.2 cm in diameter and weighed; the weight was 60 mg. They were then placed into 1.5 mL eppendorf tubes. Each tube was filled with 1 mL phosphate buffered saline (PBS) solution and sealed tightly, and then incubated in an air rotator at 100 rpm in 37 °C. At the initial time point and predetermined time intervals, 100 µL of the supernatant in the tubes was collected and fresh PBS in equal volume was added. The release of ICG-001 in the buffer was detected by high-performance liquid chromatography (HPLC) in the Biomaterials and Tissue Engineering Laboratory of Donghua University, China. The eluent used in the HPLC process is acetonitrile . A standard curve was made with gradient dilution of the ICG-001 DMSO solution from 10 to 10 thousand. The releasing experiments were performed and compared with the standard curve in triplicate every three days and completed until 30 days. The data were obtained and carefully analyzed to determine the concentration of ICG-001 released from the specimens at each immersion time point.All the animal experiments were in accordance with the guidelines for animal care. The animal protocol (SYXK 2011-0128) was approved by the animal ethics committee of Shanghai Sixth People’s Hospital, Shanghai, China. The project identification code is 14JC1492100, which is approved from 1 September 2014 by Science and technology commission of Shanghai, China.Bladder biopsies and epithelial cell harvesting were made using 12 male New Zealand white rabbits. The rabbits were pretreated with 15 mg/kg Ketamine, 2 to 3 mg/kg xylazine and 0.75 mg/kg acepromazine intramuscularly, then they were anesthetized and maintained with 2% isoflurane. A small laparotomy incision was made above the pubic symphysis to expose the bladder. A biopsy specimen with 2 × 2 cm was excised from the bladder wall, then the defect was closed with 3-0 polyglactin sutures in 2 layers. The rabbits were given 5 mg/kg enrofloxacin intramuscularly for 3 days after operation.The specimen was processed in a sterile condition. It was washed with PBS with 100 IU/mL penicillin and 100 µg/mL streptomycin. The epithelium was scraped from smooth muscle layer after using dispase type 2 enzyme (Roche) at 4 °C overnight. Then the epithelium was cut into small fractions and incubated in 0.25% trypsin solution for 15–30 min. The solution of cells were collected and cultured in 10 cm culture dish coated with 1% Type 1 rat tail collagen. Defined keratinocyte serum-free medium  with supplements was used as culture medium. Before being seeded into the scaffold, epithelial cells were identified with anti-pan cytokeratin antibody AE1/AE3 .2 of rabbit dermal tissue at abdomen was excised from one rabbit and rinsed under sterile conditions, then it was cut into small pieces and digested with collagenase type 1. The cells were cultured in 6-well plates with DMEM supplemented with 10% fetal calf serum. Then they were identified with vimentin antibody before being used .Dermal fibroblasts were used to perform the collagen inhibition assay in this study. Two cm5 cells/cm2. Cells were cultured with defined keratinocyte serum free medium (DKSFM) for 7 days before being used.To prevent waste of ICG-001 in the sterilizing process with 70% ethanol, the scaffolds were sterilized with ultraviolet for 2 h. The epithelial cells were seeded on the surface of the scaffolds with 5 × 10At days 3 and 7, cell-seeded scaffolds were rinsed with PBS to remove the non-adherent cells. Then the cells with scaffolds were fixed in 2.5% glutaraldehyde for 30 min at room temperature. Afterwards, they were dehydrated through a series of graded alcohol solutions. The drying process was conducted with the critical point dryer . The scaffolds were sputter coated with gold-palladiu (AuPd), and examined under SEM at 12 kV.Cell proliferation was tested quantitatively by using the MTT assay at day 1, 3 and 7. Cells on scaffold were incubated with MTT . After 3 h of incubation, the medium was transferred into wells of a 96-well plate. The data were read at 490 nm in a synergy plate reader.Ten thousand fibroblasts were transferred to each well of 4-well chamber slides and cultured overnight. The ICG-001 delivered scaffold was sterilized with ultraviolet for 2 h. To collect the ICG-001 medium, 2 mL of complete culture medium was used to immerse 120 mg ICG-001 delivered scaffold to get ICG-001 solution for 24 h. TGF-β1 (Life Technologies) 5 ng/mL was used to induce a phenotype change from fibroblasts to myofibroblasts and overexpress ECM according to the protocol . Four gr® 488 and 594 conjugate, Thermo Fisher Scientific, Waltham, MA, Country) for 30 min at 37 °C. The nuclei were stained with Fluoroshield Mounting Medium  with DAPI. The cells were examined with fluorescence microscopy.The primary monoclonal antibodies were anti-collagen type 1 and anti-collagen type 3 from mouse . The cells were treated with 0.2% Triton X-100 for 10 min at room temperature and incubated with the primary antibody for 60 min at 37 °C, then the cells were rinsed with PBS 3 times and incubated with primary antibody and fluorescent labeled secondary antibody (Donkey anti-Mouse IgG (heavy + light chain) Secondary Antibody, Alexa FluorAt day 3 of treating, total RNA was extracted for the quantification of collagen type 1, type 3, TIMPs, MMPs, β-catenin and α-SMA , cDNA was synthesized . TaqMan probes were applied for the quantification of the target genes. β-Actin was used as an endogenous quality control.At day 3, western blot analysis was conducted to analyze the relative expression level of collagen type 1, 3, fibronectin and α-SMA in fibroblasts treated with TGF-β1 and culture medium released from the ICG-001 delivering scaffold. The lysis buffer  was used to extract the proteins. After running on a 6% gel, proteins were transferred to nitrocellulose membranes . Membranes were blocked with Tris-buffered saline with 0.1% Tween-20 (TBST) containing 5% nonfat dry milk at room temperature, then incubated with primary antibodies at 4 °C overnight and subsequently with HRP (horseradish peroxidase)-conjugated goat anti-mouse secondary antibody for 1 h at room temperature. Anti-β-actin antibody was used as a protein loading control. The results were quantified using Quantity one and showed as the relative expression to β-actin.Twelve male New Zealand white rabbits were divided into 2 groups. Six rabbits were in group 1 and treated with non-drug scaffold with epithelial cells seeded. Group 2 were treated with an ICG-001 delivering scaffold with epithelial cells seeded. After general anesthesia with intravenous injection of pentobarbital, Foley F8 silicone catheters  were inserted into the urethra of 18 male rabbits. All surgeries were performed by an urologist. Briefly, the skin approximately 3 cm from the external urethral orifice was sectioned, and the urethra was dissected from the corpus cavernosum. Ventral urethral defects (mean length of 2.0 cm and width of 0.8 cm) were created in the bulbar urethra of rabbits. The scaffolds (length of 2 cm and width of 1 cm) were tubularized and sutured to form tubes, then sutured to the urethra defect with 6-0 absorbable polyglactin sutures. The 8F silicone catheter was left in the urethra and fixed to the glan of the rabbit with 6-0 absorbable sutures for 14 days postoperatively. The animals were observed twice on each day before catheters were removed; if the animal removed the catheter, another new catheter would be reinserted after anesthesia. Euthanization of the rabbits in the 2 groups after 3 months was planned.Retrograde urethrograms were made for the animals in both groups to assess urethral caliber before animals were euthanized.The urethras were harvested for histology analysis. Hematoxylin and eosin stain (H&E), Masson trichrome staining and AE1/AE3 immunohistology test were conducted to identify the epithelium layer, smooth muscle and collagen. The Masson staining images were used to collagen and smooth muscle analysis, and the AE1/AE3 staining images were used for epithelial analysis.p < 0.05 was considered statistically significant.Results are expressed as mean ± standard deviation. SPSS statistical software 16.0  was applied to calculate the data by one-way analysis of variance; in vitro study verified the biocompatibility and long term release fashion of ICG-001 in the scaffold. Immunofluorescence, PCR and Western blot results demonstrated that the ICG-001 delivering scaffolds are able to significantly inhibit ECM expression of fibroblasts. The results presented here using the rabbit urethral defect model provides a foundation for further study with potential clinical applications in the future.We successfully constructed ICG-001 delivering collagen/P(LLA-CL) scaffolds with high mechanical properties. The


Lactobacillus plantarum NDC 75017 on D-galactose -induced mitochondrial dysfunction in the rat cerebral cortex. Fifty rats were randomly divided into five groups (n=10 in each group). The rats in the aging model group were subcutaneously injected with 100 mg/kg D-gal and those in the protective groups were additionally orally administered L. plantarum NDC 75017 at doses of 1×108, 1×109 or 1×1010 CFU/100 mg body weight/day, respectively. The control rats were administrated an equal volume of the vehicle. Following continuous treatment for seven weeks, the learning and memory abilities and mitochondrial ultrastructure, function and adenosine triphosphate (ATP) levels were examined. The results showed that the learning and memory abilities and mitochondrial levels of ATP were significantly decreased in the D-gal-induced aging model group compared with those in the control group (P<0.01). In addition, marked changes in the mitochondrial functions and ultrastructure were observed between the groups. Seven weeks of L. plantarum NDC 75017 and D-gal coadministration significantly improved the learning and memory abilities of the rats compared with the D-gal-induced aging model group. Furthermore, the combination regime significantly improved the mitochondrial ultrastructure and functions, including the mitochondrial respiratory chain, mitochondrial membrane potential and mitochondrial permeability transition. The results revealed that the L. plantarum NDC 75017 was able to alleviate learning and memory injuries in aging rats by reducing the mitochondrial dysfunction induced by D-gal.The aim of the present study was to investigate the protective effect of  Mitochondrial dysfunction plays a major role in the process of aging and in aging-related neurodegenerative disorders. This is due to the crucial role of mitochondria in producing adenosine triphosphate (ATP), the main source of cellular energy –4. Furthet al  and medicinal plants. It has been revealed that several dietary supplements, including spinach and citrus fruits extracts, may be beneficial in protecting against age-related neurological disorders ,17. A nuet al  demonstray et al  reporteday et al . A previay et al . This isay et al . In a properties –25. In tD-gal, rhodamine 123 (Rh123), rotenone and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Sigma-Aldrich . Bicinchoninic acid protein and ATP assay kits were purchased from Wuhan Boster Bioengineering Co. Ltd.  and Beyotime Institute of Biotechnology , respectively. All other chemicals used were of the highest quality that is commercially available. The JSM25610LV transmission electron microscope used in the study was produced by Japan Electron Optics Laboratory Co., Ltd. .L. plantarum NDC 75017 were isolated from a traditional Chinese fermented yogurt . The bacteria were anaerobically grown at 30°C overnight in de Man-Rogosa-Sharpe broth . The bacterial cells were collected by centrifugation at 8,000 × g for 5 min, washed three times with phosphate-buffered saline and adjusted to 1×108, 1×109 and 1×1010 CFU/ml for oral administration to the rats.The L. plantarum once per day for 49 days (seven weeks). The rats in the control and aging model groups were only administered a vehicle  or D-gal, respectively. The treatments were as follows: Group I, 0.9% normal saline (control group); Group II, D-gal (100 mg/kg) subcutaneously ; Group III, low-dose L. plantarum  plus D-gal (100 mg/kg) ; Group IV, medium-dose L. plantarum  plus D-gal (100 mg/kg) ; Group V, high-dose L. plantarum  plus D-gal (100 mg/kg) .A total of 50 male Wistar rats (weighing 180–200 g) were obtained from the Vital River Laboratory Animal Technology Co., Ltd. . The rats were housed in separate cages and had free access to food and water for ≥1 week to acclimate prior to the initiation of treatment. The animals were housed in a limited-access animal facility where the room temperature and relative humidity were set to 22±2°C and 55±10%, respectively. Artificial lighting provided a 24-h cycle of 12-h light/12-h dark (light from 7:00 a.m. to 7:00 p.m.). All of the animal experiments were approved by the Animal Care and Use Committee of Heilongjiang Province, China. After the one-week acclimation period, the rats were randomly divided into five groups, with 10 rats in each group. The rats were orally administered 1 ml/100 g body weight of different concentrations (CFU/ml) of Spatial learning was investigated after the seven weeks of D-gal injection using the Morris water escape task according to a previous study . From thAfter seven weeks of treatment, the mitochondrial ultrastructure of the rat cerebral cortices was observed using a transmission electron microscope as previously described . A totalMitochondria were isolated from the cerebral cortices of the rats through homogenization and differential centrifugation according to the methods performed in a previous study . The proThe levels of ATP in the mitochondria were measured using the ATP Bioluminescence Assay kit (Beyotime Institute of Biotechnology) according to the manufacturers’ instructions. Briefly, the levels of ATP were determined by mixing 50 μl mitochondrial solution with 50 μl luciferase solution, which catalyzes ATP-mediated light production from luciferin. The amount of emitted light, measured using a microplate luminometer , was linearly associated with the ATP concentration.540 nm), as previously described  was measured using an F-4500FL spectrophotometer . The diluted mitochondria (0.5 mg/ml) were added to the buffer and incubated for 3 min. The fluorescence was measured again using the F-4500FL spectrophotometer. Finally, the change in Δψm was expressed by the decrease in fluorescence.Δψus study , with moMTT reduction was used to assess the activities of the mitochondrial respiratory chain. The methods and procedures utilized in the present study were identical to those of a previous study . BrieflyData are presented as the mean ± standard deviation. The data were analyzed using one-way analysis of variance followed by least significant difference post hoc tests to compare the different treatment groups. P<0.05 was considered to indicate a statistically significant difference.From the 44th day, the rats were trained for four days, following which the water maze test was carried out. The escape times of the rats in D-gal group were significantly higher (P<0.01) compared with those of the control group rats. However, the escapes times of rats in the L + D-gal, M + D-gal and H + D-gal groups were significantly lower (P<0.01) compared with those of rats in the D-gal group .L. plantarum NDC 75017 and D-gal decreased the neuronal mitochondria injury in rats with dose-dependent effects  was markedly reduced following the addition of mitochondria. As shown in ΔψThe reduction of the water-soluble tetrazolium salt MTT to formazan is regarded as an indicator of mitochondrial respiration, particularly the activity of mitochondrial succinate dehydrogenase. Compared with the control group, mitochondrial enzymatic activity was significantly lower (P<0.01) in the D-gal group, and progressive improvements were observed in the L, M and H + D-gal groups .Several theories have been proposed to explain age-related mitochondrial dysfunction. The oxidative stress theory is the most important theory of aging proposed in past few decades . The tar2+ homeostasis is the center of widespread interest in scientific studies due to its modulatory role in numerous physiological processes and its involvement in cell death (2+ uptake and release from the mitochondrial membrane via a variety of mechanisms control the local regulation of intracellular Ca2+ concentration. The mitochondrial Ca2+ dysfunction can cause MPT pore opening, leading to a change in the Δψm and resulting in mitochondrial swelling and dysfunction, and even cell death (m, mitochondrial membrane permeability and ATP production ability. These changes were alleviated with the administration of L. plantarum NDC 75017, which may be associated with its antioxidative properties.At present, mitochondrial Call death ,28,29. Cll death ,28. The L. plantarum NDC 75017 has the ability to produce high levels of GABA in vitro, which may contribute to its anti-aging and D-gal-induced mitochondrial dysfunction-alleviating abilities. The precise mechanisms for this should be investigated in further studies.GABA supplementation may activate the upstream signal survival pathways that regulate mitochondrial function, such as the phosphoinositide 3-kinase-Akt signal survival pathway . GABA alL. plantarum NDC 75017 was able to alleviate learning and memory-associated injuries in aging rats by reducing mitochondrial dysfunction induced by D-gal. This may be associated with its antioxidant and GABA-producing activities.In conclusion, the results of the present study revealed that


Barley, globally the fourth most important cereal, provides food and beverages for humans and feed for animal husbandry. Maximizing grain yield under varying climate conditions largely depends on the optimal timing of flowering. Therefore, regulation of flowering time is of extraordinary importance to meet future food and feed demands. We developed the first barley nested association mapping (NAM) population, HEB-25, by crossing 25 wild barleys with one elite barley cultivar, and used it to dissect the genetic architecture of flowering time.G). The strongest single QTL effect corresponded to the known photoperiod response gene Ppd-H1. After sequencing the causative part of Ppd-H1, we differentiated twelve haplotypes in HEB-25, whereof the strongest exotic haplotype accelerated flowering time by 11 days compared to the elite barley haplotype. Applying a whole genome prediction model including main effects and epistatic interactions allowed predicting flowering time with an unmatched accuracy of 77% of cross-validated pG.Upon cultivation of 1,420 lines in multi-field trials and applying a genome-wide association study, eight major quantitative trait loci (QTL) were identified as main determinants to control flowering time in barley. These QTL accounted for 64% of the cross-validated proportion of explained genotypic variance  contains supplementary material, which is available to authorized users. Barley is among the oldest crop species human civilization was built on. Approximately 10,500 years ago, barley was domesticated in the Fertile Crescent , presumaPpd-H1, a PSEUDO-RESPONSE REGULATOR 7 (PRR7) gene, in its center [Ppd-H1, through mediation of CONSTANS (CO), promotes the expression of the floral inducer Vrn-H3, a homolog of the Arabidopsis thaliana FLOWERING LOCUS T (FT) gene [Vrn-H2, a zinc-finger CONSTANS, CO-like and TOC1 (CCT)-domain protein (ZCCT1) acts as a repressor of Vrn-H3 [Vrn-H2, in turn, is repressed by Vrn-H1, an APETALA1 family MADS-box transcription factor [Vrn-H3 is abolished and flowering is induced. Based on its vernalization requirement, winter barley and spring barley can be distinguished. Spring barley lacks the vernalization requirement due to a deletion of Vrn-H2 [In this regard, time of flowering is expected to play a major role in future crop improvement. It is a key trait for the successful completion of a plant’s life cycle and, therefore, it has a strong impact on grain yield . Floweris center . Under lFT) gene . On the f Vrn-H3 . Vrn-H2,n factor , which if Vrn-H2 .per se [A. thaliana [Besides photoperiod and vernalization, there are also genetic mechanisms acting independently of environmental cues, so-called earliness per se . Althougthaliana ,21-23. Sthaliana ,24,25.Hordeum vulgare ssp. vulgare, Hv) and 25 highly divergent exotic barley accessions, contributing an ideal instrument to study biodiversity. The exotic donors comprise 24 wild barley accessions of H. vulgare ssp. spontaneum (Hsp), the progenitor of domesticated barley, and one Tibetian H. vulgare ssp. agriocrithon (Hag) accession. Barke was selected since it was also used as a parent of a barley high-resolution mapping population [et al. [1 plants were backcrossed to Barke and, subsequently, selfed three times  emerged as a multi-parental mapping design to investigate genomic regions with unprecedented genetic resolution and allelic variation by combining the advantages of linkage analysis and association mapping . Hence, pulation  and as apulation . The exo [et al.  to repreIn the present study we investigated the genetic architecture of flowering time in barley. For this purpose, the NAM population HEB-25 was grown from 2011 to 2013 in multi-field trials to gather data on flowering time. By combining these data with high-density SNP marker information via genome-wide association studies and genomic prediction models, we could show that flowering time in barley mainly depends on a low number of large-effect QTL and epistatic interactions.The inheritance of parental segments across the genomes of the 1,420 HEB lines was characterized through genotyping 5,709 informative, genic single nucleotide polymorphism (SNP) markers . Marker Diversity in HEB-25 was also visible phenotypically. During the seasons 2011 through 2013, HEB-25 was cultivated at the Halle University Experimental Field Station to collect flowering time data. The HEB lines flowered on average 68.1 days after sowing with a range from 51.0 to 98.9 days and a standard deviation of 6.5 days  was observed on the short arm of chromosome 2H and explained a pG of 36%. This SNP is directly located within Ppd-H1, the major determinant of photoperiod response in barley under long day condition [Ppd-H1, also the vernalization genes Vrn-H1 and Vrn-H2, as well as the floral inducer Vrn-H3 and its putative paralog HvCEN [denso [HvELF3 [For GWAS, we initially applied the multiple linear regression Model-B with step-wise selection of cofactors, as outlined in Liu et al. . Model-Bet al. , where a10 Table  and 7. TTable G, ) of 64% Table G, , affectsdetected ,42. The ondition . Seven fog HvCEN  exhibiteog HvCEN  through N [denso  and HvEL [HvELF3 ,47 as tw [HvELF3 ,48. So fPpd-H1 and Vrn-H3), exactly those SNPs revealed the highest significance within the respective QTL window  and 25 highly divergent exotic wild barley accessions. The latter were used as pollen donors. Twenty-four accessions, originating from Afghanistan, Iran, Iraq, Israel, Lebanon, Turkey, and Syria (Hordeum vulgare ssp. spontaneum), were selected to maximize the genetic diversity in HEB-25. One further accession, HID380, originating from Tibet, China, was classified as Hordeum vulgare ssp. agriocrithon (Åberg). F1 plants of the initial crosses were backcrossed with Barke as the female parent. Twenty BC1 plants per cross were subsequently selfed three times, using the single seed descent (SSD) technique to generate the next generations. The resulting BC1S3 generation consists of 1,420 individual lines, classified in 25 HEB families with 22 to 75 individual lines per family  was initiated in 2007 conducting crosses between the spring barley cultivar Barke , heterozygous and homozygous donor genotypes, respectively. In total, 1,027 monomorphic SNPs and 1,128 SNPs with high failure rates  were excluded from the dataset, resulting in 5,709 informative SNPs for further analyses.SNP genotype data were collected at TraitGenetics, Gatersleben, Germany, for all 1,420 individual BC864 SNPs . At each1S3. The subsequent seed propagation of HEB lines was based on these founder HEB plants. For Barke and the wild barley accessions leaf material from three to four plants was used to create bulked samples. The plants were cultivated in a glasshouse and 50 to 100 mg of leaf material was harvested for each sample. DNA was extracted according to the manufacturer’s protocol, using the BioSprint 96 DNA Plant Kit and a BioSprint work station , and finally dissolved in distilled water at approximately 50 ng/μl.DNA was extracted from leaf tissue of 1,420 single founder HEB plants in generation BCet al. [P-value likely indicate a tight linkage. Therefore, we assigned the position of the SNP with the lowest P-value (minimum: P < 0.001) to the non-mapped SNP under investigation. If there were more than one SNP with the same P-value, the position of the unmapped SNP was defined as the average of the minimum and the maximum position of the respective markers. In this way, all except six of the non-mapped SNPs were placed into the Comadran map.The chromosomal positions of 3,391 out of 5,709 SNPs were taken from Comadran et al. . The remThe differentiation of the HEB genotypes was based on an identity-by-state approach. Based on parental genotype information, the exotic allele could be specified in each segregating family. Thus, HEB lines that showed a homozygous exotic genotype were assigned a value of 2 and HEB lines that showed a homozygous Barke genotype were assigned a value of 0. Consequently, heterozygous HEB lines were assigned a value of 1. If a SNP was monomorphic in one HEB family but polymorphic in a second family, lines of the first HEB family were assigned a genotype value of 0 to keep a full genotype data set, which is a pre-requisite for the subsequent multiple regression analysis. For the same reason, missing genotypes were estimated applying the mean imputation (MNI) approach . For thiProc Distance, based on a simple matching comparison between the three possible genotype states across all informative SNPs. In addition, we performed principal component analysis (PCA) using R [SAS 9.4 Software  was used to evaluate genetic diversity among parents and progenies of the HEB-25 population. Genetic similarities (GS) between HEB lines and their parents and among HEB lines were calculated with  using R . First w using R .2 [2 between SNP pairs against their genetic distance. A second-degree smoothed loess curve [Proc Loess. The population-specific baseline r2 was defined as the 95% percentile of the distribution of r2 for unlinked markers [LD was calculated as r2  between 2 . For thiss curve  was fitt markers . LD decaPpd-H1 locus on chromosome 2HS we used the following primers: PP05 (forward) 5′-GTGCAAAGCATAATATCAGTGTCC-3′ and PP04 (reverse) 5′-GGCCAAAGACACAAGAATCAG-3′. These primers amplify the last two exons and three introns of Ppd-H1 covering the CCT domain that contains SNP22, the causal SNP of Ppd-H1 [et al. [For sequencing of the f Ppd-H1 . Identic [et al. .1S3 lines . Sowing occurred in single to five row plots with a length of 1.50 m and a distance of 0.20 m between rows. The number of rows per HEB line and the position inside the field trial depended on the number of available BC1S3:4 seeds. Lines with seed numbers lower than ten were planted in plots with a length of 0.50 m. In 2012 and 2013, the field trials were conducted with the selfed progenies in BC1S3:5 and BC1S3:6, respectively. Two replications per HEB line, arranged in two randomized complete blocks, were cultivated in 2012 and 2013. The plots consisted of two rows (30 seeds each) with a length of 1.50 m and a distance of 0.20 m between rows. All field trials were sown in spring between March and April with fertilization and pest management following local practice.Between 2011 and 2013, three field trials were conducted at the ‘Kühnfeld Experimental Station’ of the University of Halle to gather phenotype data on flowering time. In 2011, the field trial was conducted with selfed progenies of BC2) was estimated on an entry-mean basis. Best linear unbiased estimates (BLUEs) of flowering time were calculated for each genotype assuming fixed genotype effects.The occurrence of flowering time was recorded as days after sowing, when the first awns were visible (BBCH49 ) for 50%et al. [Proc Glmselect in SAS and minimizing the Schwarz Bayesian Criterion [BON-HOLM < 0.05. Additive effects for each SNP were estimated based on regression across but also within families. Significant marker trait associations were grouped to a singled QTL if the significant SNPs were linked by less than 5 cM and revealed additive effects of the same direction, i.e. both exotic alleles increased or decreased flowering time. In addition, a two-dimensional epistasis scan was carried out to identify pairwise marker interactions. For this, the GWAS Model-B was extended to cover a second main SNP effect and the interaction effect between the two SNPs.For GWAS, we applied Model-B as outlined in detail by Liu et al. . This moet al. . It is bet al. . Cofactoriterion . The genriterion , excludiriterion  was usedPpd-H1. We used the same GWAS procedure with cofactor selection as mentioned before. However, bi-allelic SNPs covering the region of Ppd-H1 were replaced by a qualitative variable containing the defined Ppd-H1 haplotype. BLUEs were determined for each haplotype. Subsequently, pairwise comparisons between all haplotype BLUEs were performed using the Tukey-Kramer [A haplotype-based association mapping test was implemented in HEB-25 to test for effects of haplotypes at y-Kramer  multipleBased on BLUEs of the 1,420 HEB genotypes, two approaches for genomic prediction were applied considering additive effects: ridge regression best linear unbiased prediction (RR-BLUP ) and Bayn be the number of genotypes, m be the number of markers and l be the number of environments. The RR-BLUP model has the form y = 1nμ + Xg + e, where y is the vector of BLUEs of flowering time scores for all HEB genotypes across environments, 1n denotes the vector of 1’s, μ is the overall mean, g is the vector of marker effects , X is the corresponding design matrix and e is the residual term. In the model we assumed that Let quations .g from all later cycles were averaged to obtain estimates of the marker effects.The basic model of BayesC π is the same as RR-BLUP. However, all parameters are treated as random variables in a Bayesian framework. First, we defined the prior distributions as G), we estimated the effects of the significant peak markers within the estimation set and predicted the genotypic value of the lines in the test set [G as the squared Pearson product–moment correlation between predicted and observed genotypic values in the test set standardized with the heritability. The mean pG in 100 cross-validation runs  was taken as the final record. In addition, the number of significances for each SNP was cumulated across all runs and is referred to as QTL detection rate.The accuracy of the prediction of flowering time by GWAS and the two genomic prediction approaches were evaluated using five-fold cross-validations . In eachtest set . We thenG was calculated as in GWAS.For genomic prediction we estimated the effects for all markers using the estimation set and predicted the genotypic value of the lines in the test set. The cross-validated py is the vector of BLUEs of flowering time for all HEB genotypes, 1n denotes the vector of 1’s, μ is the overall mean, gi is the main additive effect of the i-th marker, Xi is the vector of marker indices, fjl is the epistatic effects of the j- and the l-th marker, XjXl is the point-wise product of the two vectors Xj and Xl, and e is the vector of residual terms. Note that in the third term of the right hand side of the formula, the sum is not taken over all pairs of markers but only pairs of markers exhibiting a significant additive effect in the GWAS study performed previously. Hence, in different cross-validation runs, different pairs of markers were considered in the model. The model assumptions are similar to the usual RR-BLUP, except treating additive and epistatic effects separately. We assumed PG is the cross-validated proportion of explained genotypic variance for genomic prediction, obtained previously by the RR-BLUP, only considering additive effects, m is the number of markers, p is the number of pairs of markers having significant additive effect. Therefore the penalty parameter λ is different for additive and epistatic effects.We extended the RR-BLUP based on main effects to model also epistasis for markers with significant main effects in the GWAS. The model is G in the same way as outlined above.Using the above extended model, for each cross-validation run we estimated the additive effects of all markers and epistatic effects of all pairs of markers exhibiting significant additive effects in GWAS using the estimation set. Then we predicted genotypic values of the lines in the test set and calculated the pPpd-H1 haplotypes and GWAS, and all other supporting data are provided as additional files.Raw data, including data on SNPs,


To assess the image quality of aorta obtained by dual-source computed tomography angiography (DSCTA), performed with high pitch, low tube voltage, and low iodine concentration contrast medium (CM) with images reconstructed using iterative reconstruction (IR).One hundred patients randomly allocated to receive one of two types of CM underwent DSCTA with the electrocardiogram-triggered Flash protocol. In the low-iodine group, 50 patients received CM containing 270 mg I/mL and were scanned at low tube voltage (100 kVp). In the high-iodine CM group, 50 patients received CM containing 370 mg I/mL and were scanned at the tube voltage (120 kVp). The filtered back projection (FBP) algorithm was used for reconstruction in both groups. In addition, the IR algorithm was used in the low-iodine group. Image quality of the aorta was analyzed subjectively by a 3-point grading scale and objectively by measuring the CT attenuation in terms of the signal- and contrast-to-noise ratios . Radiation and CM doses were compared.The CT attenuation, subjective image quality assessment, SNR, and CNR of various aortic regions of interest did not differ significantly between two groups. In the low-iodine group, images reconstructed by FBP and IR demonstrated significant differences in image noise, SNR, and CNR (p<0.05). The low-iodine group resulted in 34.3% less radiation (4.4 ± 0.5 mSv) than the high-iodine group (6.7 ± 0.6 mSv), and 27.3% less iodine weight (20.36 ± 2.65 g) than the high-iodine group (28 ± 1.98 g). Observers exhibited excellent agreement on the aortic image quality scores (κ = 0.904).CT images of aorta could be obtained within 2 s by using a DSCT Flash protocol with low tube voltage, IR, and low-iodine-concentration CM. Appropriate contrast enhancement was achieved while maintaining good image quality and decreasing the radiation and iodine doses. Multislice computed tomography angiography (CTA) has become the preferred method to assess aortic diseases –6. HowevReducing the iodine concentration would help to avoid contrast-induced acute kidney injury (CI-AKI) in at-risk patients because the probability of CI-AKI is mainly determined by the amount of delivered iodine –14. CadeTo the best of our knowledge, no previous report has studied the image quality of the aorta that is obtained by using low-iodine CM and IR during aortic CTA in the Flash Spiral scan mode. Therefore, the goal of this study was to assess the quality of images of the whole aorta obtained by CTA when using a combination of low-iodine CM and scanning with high pitch, low tube voltage, and IR techniques.2 (range: 17.7–29.6). Reasons for referral included suspected aortic disease (n = 20), postoperative follow-up after thoraco-abdominal vascular surgery (n = 25), endovascular aneurysm repair (n = 15), endovascular aortic dissection repair (n = 30), and follow-up examination of conservatively treated aortic aneurysm (n = 10). General exclusion criteria for contrast-enhanced CT were patients with renal insufficiency (serum creatinine > 1.5 mg/dL), BMI > 30 kg/m2, history of allergic reaction to CM, untreated hyperthyroidism, and women who were pregnant or nursing. Age, sex, height, and body weight of all patients were recorded for further analysis. The study was approved by Beijing Anzhen Hospital Ethics Committee, and written informed consent was obtained from all patients.One-hundred patients  who were referred for noninvasive whole-aortic DSCT angiography were included in this study. Patients had a mean age of 54.3 ± 15.7 years (range: 18–88 years) and a mean body mass index (BMI) of 24.4 ± 2.6 kg/m2 and BMI between 25 kg/m2 and 30 kg/m2), and each group was further divided into subgroups (low-iodine group and high-iodine group). The low-iodine group (n = 50) received Iodixanol 270 as CM . The high-iodine group (n = 50) received Iopamidol 370 as CM .Patients were randomly assigned to one of two groups, according to the iodine concentration of the CM. The 100 patients was first divided into two groups with respect to the range of BMI . All scans were performed in a cranio-caudal direction with a prospective electrocardiogram (ECG)-triggered Flash protocol. Contrast-enhanced scans were performed from the thoracic inlet to the pubic symphysis. All CT imaging data were acquired while the patient held his or her breath in deep inspiration, to eliminate respiratory motion artifacts. Scanning parameters for both groups were as follows: slice collimation of 128 × 0.6 mm with a z-flying focal spot, gantry rotation time of 280 ms, pitch of 3.2, and tube voltage of 100 kV (low-iodine group) or 120 kV (high-iodine group).CM was injected with an 18- gauge needle through the right antecubital vein by a dual-syringe power injector. A test bolus of 15 mL of CM followed by 30 mL of saline was used to evaluate the scan delay during the acquisition of a series of dynamic low-dose monitoring scans  at the middle of the descending aorta. Regions of interest (ROIs) were placed within the descending aorta to calculate enhancement over time. Monitoring scans  began to be acquired 5 s after the start of the injection. The optimal scan delay time was calculated by adding the peak enhancement time from the monitoring scan to 10 s. The actual image was acquired by using 1 mL of CM per kg body weight, followed by 30 mL of saline solution. The injection rate of CM and saline solution was 4 mL/s for all subjects.In both the low- and high-iodine groups, CTA images were reconstructed by a conventional filtered back projection (FBP) algorithm with a medium smooth kernel designed for cardiac imaging (B26f). In the low-iodine group, images were also reconstructed by a sinogram-affirmed IR algorithm  with the corresponding vascular kernel (I26f). With the IR algorithm, five adjustable strength settings (strength 1–5) were available for adaptation of the noise model (SAFIRE). As recommended by manufacturer, a medium strength of 3 was used.In both groups, transverse images were reconstructed with a slice thickness of 1 mm in 1-mm increments. Patient information was removed from all images, which were transferred to an external workstation  for further image analysis. Using the axial data, two cardiac radiologists with more than 8 years of experience in cardiac imaging reconstructed the images by volume rendering technique, maximum intensity projection, and multiplanar reconstruction .2, were drawn on three consecutive transverse sections. When the vessel area was less than 100 mm2, the ROI was drawn to encompass the entire contrast-enhanced aortic lumen. Care was taken to avoid including vessel walls, emboli, calcified plaques, areas of stenosis, or motion artifacts in the ROIs. The mean attenuation (in Hounsfield Unit [HU]) and standard deviation (SD) in each ROI on three consecutive sections were calculated for each target structure , and iliac artery bifurcation . Furtherre Figs. .aorta and SDaorta, respectively) and the paraspinal muscle tissue  were recorded. Each parameter was calculated three times, and the mean value was used in the SNR and CNR calculations. Noise ratios were determined by the following equations: SNR = SIaorta – SImuscle/SDaorta and CNR = SIaorta – SImuscle/SDmuscle [A single reader independently calculated the SNR and CNR in the aortic images. Means and SDs of attenuation of the contrast-enhanced vessel lumen  [vol) and dose length product (DLP) were recorded during the scans. The estimated effective dose was derived from the DLP by using the equation Effective dose = DLP × k, where k is the conversion coefficient for the thoraco-abdominal region (k = 0.017 mSvmGy-1cm-1) [The CT dose index volume (CTDIy-1cm-1) , 22. ThePatients were asked to rate their discomfort, in terms of pain at the injection site, sensations of cold or heat in the injected vein, and other discomfort, immediately and 15 to 20 min after injections. Scores were reported verbally on a scale from 1 (severe discomfort) to 10  . All advt-tests for normally distributed data or Mann-Whitney U tests for non-normally distributed data. Independent t-tests were performed to analyze differences between two groups regarding attenuation values, image noise, and radiation doses. The Mann-Whitney U test was used to detect differences in the subjective evaluation of image quality between the two groups. Cohen’s kappa statistic (κ) was calculated to determine the interreader agreement in the assessment of image quality, where κ > 0.81 indicated excellent, κ = 0.61–0.80 indicated good, κ = 0.41–0.60 indicated moderate, κ = 0.21–0.40 indicated fair, and κ < 0.20 indicated poor agreement. A P-value < 0.05 was considered statistically significant for all data analyses.Statistical analysis was performed with the SPSS software package . Continuous variables were expressed as mean ± SD and were analyzed by independent High-pitch CTA was successfully performed in the 100 patients with suspected aortic disorders. Patient demographics and CTA acquisition characteristics are shown in P < 0.05), but no significant difference in the mean aortic attenuation  in the mean aortic attenuation of ascending aorta root and aorta arch, but no significant difference in SNR, CNR and the mean aortic attenuation of descending aorta at L1and iliac artery bifurcation. No significant difference was found in the subgroups by BMI between 25 kg/m2 and 30 kg/m2 except the SNR of the ascending aorta root (P = 0.917). Interreader agreement was excellent for both groups (κ = 0.904).There were no significant differences in mean aortic attenuation, SNR, or CNR between the low- and high-iodine groups of the aorta and aortic branches. However, image noise differed significantly between the groups of the aorta and aortic branches Tables . In the enuation . Of the rta root . Subjectvol and DLP were significantly higher in the high-iodine group compared to the low-iodine group (P < 0.001). The iodine weight and iodine delivery rate were lower in the low-iodine compared to the high-iodine group (P < 0.001).The CTDIne group . A compaThere were no cases of contrast extravasation in any patient. In the low-iodine group, severe, moderate, and mild sensations of heat during the scan were reported by 0 (0%), 7 (14%), and 40 (80%) patients, respectively. In the high-iodine group, these values were 20 (40%), 18 (36%), and 12 (24%) patients, respectively. Three patients (6%) in the low-iodine group experienced sensations of cold, compared to no patients in the high-iodine group.In the low-iodine group, 5 (10%) patients experienced pain. In this group, 2 patients had moderate adverse reactions (rash and vomit), which occurred 1 min and 6–8 h, respectively, after CM injection. All adverse symptoms were resolved appropriately. No delayed adverse reactions occurred in the high-iodine group.Since the introduction of multidetector CT technology, CTA has become a commonly performed and routine tool to evaluate diseases of the aorta and its major branches . VariousLowering the tube voltage has the additional advantage of offering higher attenuation levels for iodinated CM at lower X-ray tube voltages because of a greater photoelectric effect and decreased Compton scattering . The inhIt is generally accepted that improved vascular visualization can be achieved by increasing the injection rate or iodine concentration of the CM , 31–32. Low-tube-voltage CT scanning has some limitations, including increased image noise due to low photon flux  and degrThe results of this study showed that it is possible to achieve diagnostic image quality by using a strength level of 3 in SAFIRE with a low tube voltage (100 kVp). In the low-iodine group, using the IR algorithm reduced image noise and improved the SNR and CNR compared to the FBP algorithm. Image noise was slightly higher in the high-iodine group compared to the low-iodine group, although SNR and CNR did not differ significantly between the two groups. These findings may suggest that a SAFIRE strength level of 3 reduces the noise and increases the SNR or CNR. When combined with a low-iodine CM and low tube voltage, IR can effectively suppress image noise and retain image quality. Moreover, the increased image noise does not necessarily result in diminished subjective image quality, because the increased attenuation of the iodine-containing arterial system and the high attenuation difference between the arterial system and surrounding tissues can partially offset the higher image noise.The major advantage of lowering the tube voltage is to reduce the radiation dose. However, high pitch and IR are also effective approaches that are increasingly used to reduce the radiation dose. Liu et al. and Bolen et al. , 39 showStudies have demonstrated that IR could increase image quality and reduce the effective radiation dose compared to FBP , 22–23. This study has several limitations. First, this study involved a relatively small number of patients (only 50 per group). Although no significant differences in objective or subjective image quality were detected, these results should be confirmed in a larger clinical cohort. Second, the diagnostic accuracy to detect aortic disease was not evaluated. The aim of the study was to assess the performance of CMs with different iodine doses in terms of image quality alone, not diagnostic accuracy. Third, only a single IR level was used in the low-iodine group. Further evaluation of different IR strength levels within each group should be performed. Lastly, we did not compare the reconstruction time between IR and conventional FBP. IR requires more time than a standard FBP reconstruction, which may influence the clinical utility.High-pitch, low-kilovoltage dual-source CT angiography is feasible in patients with suspected aortic diseases. Iterative reconstruction with SAFIRE appears to complement low-iodine CTA protocol acquired using a low voltage and high pitch technique with resultant 34% reduction in radiation dose, but with significant improvements in image quality.